Adenosine 3′,5′-monophosphate-dependent protein kinase of bovine tracheal smooth muscle

Biochimica et Biophysiea Acta, 302 (1973) 267-28I © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s

BBA 66848 ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE OF B O V I N E T R A C H E A L SMOOTH MUSCLE

H O W A R D SANDS, T H E O D O R E A. M E Y E R AND H O W A R D V. R I C K E N B E R G a

Division qf Research, National Jewish Hospital and Research Center and aDepartment of Biophysics and Genetics, University of Colorado School of Medicine, Denver, Colo. (U.S.A.) (Received October 2nd, 1972)

SUMMARY

An adenosine 3',5'-monophosphate-dependent protein kinase (ATP :protein phosphotransferase, EC 2.7.1.37) from bovine tracheal smooth muscle was purified partially and its properties studied. Histone was employed as phosphate acceptor; the apparent Km for histone was 5o/~g/ml and was not affected by adenosine 3',5'monophosphate (cyclic AMP). The apparent Km for ATP (I.4. Io-s M at IO mM Mg 2+) was also unaffected by cyclic AMP. Cyclic AMP increased the V of the phosphorylation of histone with an apparent Km cyclic AMP of 2. 5 "10 -8 M and a p H optimum of p H 6.5. Activation of the protein kinase by cyclic nucleotides other than cyclic AMP occurred only at unphysiologically high concentrations and the enzyme appeared to be specific for ATP as phosphate donor. A divalent metal ion was required for the reaction ; the apparent Km for Mg ~+ was 2.5" IO-8 M. Incubation of the partially purified protein kinase (mol. wt approx. 144 500) with cyclic AMP, followed b y sedimentation through a sucrose gradient containing cyclic AMP, resulted in the formation of two subunits. A catalytic subunit, the activity of which was not stimulated by cyclic AMP, had a mol. wt of approximately 50 ooo ; a cyclic AMP-binding subunit, devoid of catalytic activity, had a mol. wt of approx. 88 500.

INTRODUCTION

Epinephrine, fl-sympathomimetic drugs, and theophylline are used clinically to produce relaxation of bronchial and tracheal smooth muscle during asthmatic attack. They have also been shown to produce bronchiolar and tracheal relaxation in vitro1,2. I t is now assumed that the//-receptor effects of the catecholamines are mediated by stimulation of the activity of adenyl cyclase, and that theophylline inhibits the cyclic AMP phosphodiesterase resulting in an increase in intracellular levels of cyclic AMP. Cyclic AMP and its dibutyryl derivative have been shown to produce relaxation of the smooth muscle of the trachea 8, intestinO, 5 and blood vessels 6. The mechanism by which cyclic AMP produces relaxation of smooth muscle is not known. The mode of action of cyclic AMP is now understood in several systems wher(

268

H. SANDS et al.

interaction of the cyclic nucleotide with protein kinases occurs. For example, the stimulation of glycogenolysis by drugs which exert their effect on tile /7-receptor is mediated by a cyclic AMP-dependent protein kinase. This enzyme catalyzes the phosphorylation, and concomitant activation, of phosphorylase kinase v. Conversely, the activity of glycogen synthetase is inhibited by phosphorylation also catalyzed by a cyclic AMP-dependent protein kinase8, ~. The occurrence of protein kinases is widespread and has been reported from a variety of tissues as well as phyla, including mammals 1°-~6, invertebrates ~7, bacteria ~s and viruses 19. The ubiquitous occurrence of cyclic AMP-dependent protein kinases in tissues in which cyclic AMP is the presumed second messenger led to the proposal that a majority of tile effects produced by cyclic AMP are mediated by activation of protein kinases l~. We propose that one step in the production of fl-receptor-stimulated relaxation of smooth muscle is the activation of a cyclic AMP-dependent protein kinase. This paper describes the pertinent properties of a protein kinase partially purified from bovine bronchial and tracheal smooth muscle. METHODS

Materials Bovine tracheae (from a point below the larynx and including the primary bronchi) were obtained within 0.5 h of slaughter of animals and packed in ice for transport to the laboratory. Smooth muscle layers of the trachea and bronchi were carefully stripped off, cut into small pieces, frozen in dry ice, and stored at --40 °C until purification was started. Cyclic AMP, cyclic IMP, cyclic GMP, cyclic UMP, cyclic CMP, cyclic 2',3'-AMP, ATP, UTP, G T P ' , casein, protamine, bovine serum albumin, tricalcium phosphate gel, lactate dehydrogenase were purchased from Sigma, and calf thymus mixed histones from Schwarz-Mann. DEAE-cellulose (DE-52) was obtained from Reeves Angel and Biogel P-3oo from Biorad. Membrane filters, Type B-6 (0.45/,m), were purchased from Schleicher and Schuell. Carrier-flee 32p in dilute HC1 was obtained from ICN.

Pr@aratio~ of [V-3~p]A TP [)~-32p]ATP was prepared from carrier-free 32p using the method of Glynn and Chappell ~°.

Assay of protein kinase activity Protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37 ) activity was measured at p H 6.5 and 30 °C in an 0.3 ml reaction mixture containing the following : 50 mM sodium glycerol phosphate, IO mM NaF, 2 mM theophylline, 3-3 mM ethylene glycol bis-(/5-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), IO mM MgC12, 3-3/~M LB2p~ATP (containing approx, lO 6 cpm); histone, when present, was used at IOO #g and cyclic AMP at lO -6 M. The reaction was started by the addition of ATP and terminated after 7 min by the addition of 0.2 ml of a mixture containing 3O~o trichloroacetie acid, o.I mM KH2PO 4 and I mM non-radioactive ATP. The precipitate was collected on a B-6 membrane filter, washed with 50 ml of 5Yo trichloroacetic acid-o.i mM K H 2 P Q , and the radioactivity on the filter was determined in a liquid * Un less s t a t e d o t h e r w i s e Cyclic n u c l e o t i d e m e a n s Cyclic n u c l e o s i d e 3', 5 ' - m o n o p h o s p h a t e .

SMOOTH MUSCLE PROTEIN KINASE

269

scintillation spectrometer. The scintillation fluid was toluene containing o.48% 2,5diphenyloxazole-o.oo6% 1, 4 bis-E2-(5-phenoxazole)j-benzene. Routinely, values for the radioactivity representing the phosphorylation of endogenous protein were subtracted from those obtained in the presence of histone (or other exogenous substrates) when determining the activity of protein kinase with respect to exogenous substrates. The specific activity of the protein kinase is expressed as pmoles of 3~p incorporated during 7 rain per mg of protein. All assays were performed in duplicate and in the range in which activity was a rectilinear function of protein concentration. The reason for the use of the unphysiologically low concentration of 3-3 #M ATP was the difficulty in obtaining sufficiently high specific radioactivity, when physiological concentrations of ATP were employed. However the time course of the phosphorylation with histone was rectilinear with time even at the low concentration of ATP (Fig. 3). The rate of histone phosphorylation when IOO #M ATP was employed was approximately 5-fold higher than the rate at 3.3 #M ATP.

Sucrose density gradient centrifugation The method of Martin and Ames ~1 was employed in studies involving sucrose density centrifugation. Centrifugation was conducted in a Beckman SW39 rotor at 39 ooo rev./min, 5 °C, and for 28 h. Linear sucrose gradients (5-22%) in 0.02 M TrisHC1 (pH 7.4), o.I M KC1, 0.005 M MgC12, and 0.006 M 2-mercaptoethanol (Buffer A) in the presence or absence of lO -6 M cyclic AMP were used. The Biogel P-3oo fraction (24o-28o#g) in a volume of 0.2 ml was layered over the gradient. Fractions of approximately o.I ml were collected from the top of the tubes using an Isco gradient fractionator and assayed for protein kinase activity in the presence or absence of lO -6 M cyclic AMP. Horse hemoglobin (tool. wt 64 ooo) and rabbit muscle lactate dehydrogenase (tool. wt 142 ooo) were used as standards for the molecular weight calculations ~1.

Measurement of binding of cyclic A M P In order to identify, and to determine the molecular weight of, the cyclic AMPbinding component of the protein kinase, cyclic I3HIAMP ( i i o #Ci, 14 Ci/mmole) was introduced into the gradient in addition to lO -6 M unlabeled cyclic AMP. Aliquots (o.oi ml) of each fraction collected were placed on a B-6 membrane filter and washed with 5 ml of Buffer A. The radioactivity remaining on the filter, representing cyclic AMP bound to protein, was determined by liquid scintillation counting using Bray's solution 22.

Other methods Protein was determined according to the method of Lowry et al. 23 using bovine serum albumin as standard. Lactate dehydrogenase was determined by the method described in the Worthington Enzyme Manual 24. RESULTS

Partial purification of cyclic A MP-dependent protein kinase Preparation of crude homogenate. Frozen bronchial and tracheal smooth muscl~ (12oo-15oo g) was thawed and homogenized in 3 vol. of 4 mM E D T A (pH 7.0) at

H. SANI~S et al.

270

I6 ooo rev./min for 3-4 min in a Servall Omni-Mixer. The homogenate was centrifuged at 15 ooo × g for 3o min. Homogenization and all subsequent manipulations were carried out at 4 °C. Acid precipitation. The p H of the supernatant solution was brought to 4.8 by the dropwise addition of I M acetic acid with constant stirring. The precipitate was removed by centrifugation and the p H of the supernatant adjusted to p H 6. 5 by the dropwise addition of I M, p H 7, potassium phosphate buffer, Fractionation with (NH4)~SO 4. The supernatant was then saturated to 5o% with respect to (NH4)2SO 4 by the addition of 325 g/1 of solid (NH4)2SO 4. The precipitate containing the protein kinase was collected by centrifugation, redissolved in i o o 200 ml of 5 mM potassium phosphate, 2 mM EDTA, p H 7.0 (Buffer B) and dialyzed against two 4-1 volumes of the same buffer for 16 h. After dialysis, the solution was centrifuged and a small amount of precipitate discarded. Fractionation with calcium phosphate gel. 20 g (wet weight) of calcium phosphate gel equilibrated with Buffer B were added to the supernatant solution. The suspension was stirred continuously for 30 rain, then centrifuged at 5000 × g for 15 min. The gel was then suspended and stirred for 30 min in each of the following series of phosphate buffers composed of dibasic and monobasic potassium phosphate (9:I, v/v) containing I mM EDTA: 15o ml of 0.05 M, 40-5 ° ml of 0.05 M, 40-5 ° ml of o.I M, 40-50 ml of 0.2 M, and 40-50 ml of 0.3 M. After each 30 min of stirring, the gel was collected by centrifugation at 5000 × g for 15 min. The enzyme was eluted in the last two or three final washes. These wash fluids were combined and dialyzed against two 4-1 volumes of Buffer B for I6 h. DEAE-cellulose chromatography. The solution containing the enzyme was applied to a column of DEAE-cellulose (DE-52) equilibrated with 5 mM Tris-HC1, p H 7.5 (columns of either 0. 9 cm × 15 cm or 2.6 cm x 3.8 cm gave the same elution

2.0 1.8 1

DE-S2

10 9

/P'~ /

I~

/~I

/Gradient

1.0

i

~

5 ~

I

0.8

0.6

2 'o

0.2

/ 1

5

9

13

17

1 21

25

29

33

37

41

4.5

49

Fraction

Fig. ~. C h r o m a t o g r a p h y of b o v i n e s m o o t h m u s c l e p r o t e i n k i n a s e on D E A E - c e l l u l o s e . A p p r o x i m a t e l y 280 mg of p r o t e i n o b t a i n e d f r o m t h e c a l c i u m p h o s p h a t e gel were a p p l i e d t o a c o l u m n of D E A E - c e l l u l o s e (o.9 c m × 15 cm), e q u i l i b r a t e d w i t h 5 mM T r i s - H C l - i mM E D T A , p H 7-5. The c o l u m n w a s e l u t e d w i t h a l i n e a r g r a d i e n t of 5 mM to o. 5 M T r i s - H C l - i mM E D T A a t p H 7.5A c t i v i t y was d e t e r n l i n e d in t h e presence of cyclic A M P a n d h i s t o n e ; t h e r e w a s no c o r r e c t i o n for t h e p h o s p h o r y l a t i o n of e n d o g e n o u s protein. The t o t a l g r a d i e n t v o l u m e w a s 3oo ml. F r a c t i o n s 29 3I were pooled.

271

SMOOTH MUSCLE PROTEIN KINASE 0.7

~,~

P-300

0.6

E c o

I I

0.5

10

0.4

8::

o.3

0.2

4

0.1

2

o

x

4

0

8

12 16 20 24 28 32 36 Fraction No,

Fig. 2. C h r o m a t o g r a p h y of bovine s m o o t h muscle protein kinase Biogel P-3oo. A p p r o x i m a t e l y 3 ° m g of protein were applied to a c o l u m n of P-3oo (2. 5 cm × 35 cm) which h a d been equilibrated w i t h 5 mM p o t a s s i u m p h o s p h a t e a n d 2 mM E D T A , p H 7.o. The v o l u m e of the protein solution was reduced to 4 ml b y v a c u u m dialysis, Activity was determined in the presence of cyclic AMP and histone; there was no correction for t h e p h o s p h o r y l a t i o n of endogenous protein. Approxim a t e l y I o - m l fractions were collected. F r a c t i o n s 17-I 9 were pooled.

pattern; however, the latter had a considerably faster rate of flow). The enzyme was eluted at p H 7.5 with 300 ml of 0.005 M to 0. 5 M linear Tris-HC1 gradient containing I mM EDTA. All protein kinase activity adhered to the DEAE. A typical elution pattern is shown in Fig. I. The enzyme was eluted as a single major peak; fractions with protein kinase activity were pooled and dialyzed against two 4-1 volumes of Buffer B. A minor peak of kinase activity was seen in several preparations. Further studies on the minor peak of activity were not conducted. Chromatography on Biogel P-3oo. The preparation resulting from the preceding step was reduced in volume to 4-7 ml b y vacuum dialysis and placed on a 2.5 cm × 35 cm column of Biogel P-3oo which had been equilibrated with Buffer B. The enzyme was eluted with Buffer B. A typical elution pattern is shown in Fig. 2. The enzyme was eluted slightly behind the main protein peak which occurred at approximately the TABLE I PURIFICATION

OF

SMOOTH

MUSCLE

PROTEIN

KINANE

Protein kinase was purified as described in t h e text. Specific activity and total activity were determined in the presence of histone and cyclic AMP after correction for p h o s p h o r y l a t i o n of endogenous protein.

Fraction

Protein (rag)

Specific activity"

Purification (-fold)

Total activity X

Crude h o m o g e n a t e p H 4.8 s u p e r n a t a n t o - 5 o % s a t u r a t e d (NH4)2SO 4 precipitate After calcium p h o s p h a t e gel D E A E cellulose Biogel P-3oo

Yield (%)

10 -3t

3 ° o28 15 830

162 240

-1.5

4865 3799

ioo 78.1

3 9 °6 288 31 8

374 2662 6045 io 51o

2-3 16 37 65

1461 767 188 84

3 °.0 15.8 3.9 1.7

* pmoles of 3,p incorporated in 7 m i n per mg of enzyme. t pmoles of 32p incorporated in 7 min.

272

H. SANDS

dt Cl]

/

180 160 140

280

120

240

100

200

80

160

o v mc o~ o E o.

6O

m

40

E D.

i

2oi 0

120 80 40

4

8

12

Time (min.)

16

20

I 10

20

30 Protein

40

~cAMPI

'1

50

70

60

(l~g)

Fig. 3. Time course of phosphorylation of histone by smooth muscle protein kinase in either th( presence or absence of Io -e M cyclic AMP. Conditions of incubation were as described unde: Methods except for the indicated time of incubation. The reaction mixture contained 8.4 pg o protein from the P-3oo fraction. Fig. 4. Effect of the concentration of enzyme on activity in either the presence or absence of lO-8 ~' cyclic AMP. Conditions were as described under Methods except for the variation of the concen tration of enzyme. The reaction mixture contained 8.4 pg of protein from the P-3oo fraction. void volume of the column. The fractions c o n t a i n i n g p r o t e i n kinase a c t i v i t y wet( pooled a n d frozen in 1-1. 5 ml volumes a n d stored at - - 4 ° °C. No change in enzymic a c t i v i t y was seen over a t w o - m o n t h period. A s u m m a r y of the purification procedur( is presented in Table I; similar results were o b t a i n e d in three separate preparations Tile activities shown in Table I were observed in the presence of IO-6 M cyclic AMP The s t i m u l a t i o n produced b y cyclic AMP was similar t h r o u g h o u t the purification. Ge electrophoresis of 80 #g of the P-3oo fraction o n o.I°//o sodium dodecyl sulfate, Io°/ acrylamide gels showed the presence of five m a j o r protein bands.

Properties of protein kinase from tracheal smooth muscle Time course of phosphorylation of histone. The p h o s p h o r y l a t i o n of histones al 30 °C was rectilinear with time for lO-12 m i n in either the presence or absence of i o ' M cyclic AMP (Fig. 3)- A n i n c u b a t i o n time of 7 rain was chosen for the s t a n d a r d assa 3 of protein kinase activity. It should be stressed here t h a t although histones are c o n v e n i e n t substrate for the assay of protein kinase activity, t h e y p r o b a b l y have littk physiological significance with respect to a role of a smooth muscle p r o t e i n kinase. Effect of varying concentration of enzyme. The d a t a presented in Fig. 4 show thal u n d e r s t a n d a r d conditions the p h o s p h o r y l a t i o n of histones was rectilinearly p r o p o r tional to the c o n c e n t r a t i o n of purified enzyme protein up to a c o n c e n t r a t i o n of 15/t~ of protein per t u b e (50 ¢tg/ml) in either the absence or presence of lO _6 M cyclic AMP The following assays for the characterization of the enzyme were carried out with 8.z ttg of p r o t e i n / t u b e . pH optimum. Fig. 5 illustrates the effect of p H on the p h o s p h o r y l a t i o n of tile

273

SMOOTH M U S C L E P R O T E I N K I N A S E

6070

o

o

p

4o

30 E

20

[

0. I0

-cAMP

~

o!

5

•

-

"

"

L

I

I

6

7

8

pH

Fig. 5. Effect of p H on p h o s p h o r y l a t i o n of h i s t o n e by s m o o t h m u s c l e p r o t e i n kinase in either the presence or absence of lO -6 M c y c l i c A M P . C o n d i t i o n s were as described under M e t h o d s except for the v a r i a t i o n in p H . The reaction m i x t u r e c o n t a i n e d 8, 4 / , g of p r o t e i n from t h e P-3oo fraction.

histone. Maximal activity in the presence of lO -6 M cyclic AMP was in the range of pH 6- 7. In the absence of cyclic AMP a braoder pH optimum of 6.5-8.0 was observed ; cyclic AMP stimulated maximally at pH 6.5. Substrates of protein kinase. The identity of the "natural" substrate of smooth muscle protein kinase is not known at present. Table II shows that of the proteins tested as substrate, histone was the most effective in both the presence and absence of IO-~ M cyclic AMP. Protamine was phosphorylated at about 40% of the rate of histone. Cyclic AMP had a similar stimulatory effect on the phosphorylation of histone and protamine. Casein and bovine serum albumin were phosphorylated at only a fraction of the rate observed with histone. Because of the low extent of phosphorylation, it was difficult to quantitate the stimulatory effect of cyclic AMP on the phosphorylation of casein and bovine serum albumin. Apparent Km of protein kinasefor histone. In view of the relative effectiveness of histone as a substrate and its use as a substrate in the purification and characterization of the enzyme, it was of interest to determine the apparent Km of the protein kinase for histone (Fig. 6), Fig. 6A shows that the phosphorylation of histones was linear with increasing histone concentration to approximately IOO #g (33o #g/ml)

TABLE

II

SUBSTRATE

SPECIFICITY

OF S M O O T H M U S C L E P R O T E I N

KINASE

C o n d i t i o n s were as described u n d e r E x p e r i m e n t a l P r o c e d u r e s except for the v a r i a t i o n of s u b s t r a t e The reaction m i x t u r e c o n t a i n e d 8. 4 / ~ g of p r o t e i n f r o m t h e P-3oo fraction. Substrate Ioo /,g

Histone Protamine Casein Bovine serum albumin

pmoles 32p incorporated

-- cyclic A M P

+ cyclic A M P

27-4 i 2.6 o.2

I o 1.9 42.4 2. i 0.8

o.o

Stimulation by cyclic A M P (-fold)

3.7 3.6

H. SANDS eta/.

274

~o .oo~

120

/ r

100 600

80 60

~ook

•

2

0

/

,40 o

2

o

0

40

~ 80

120

- -

160

200

0

~

10

20

Hislone (l~g)

30

1

40

5o

60

XlOO(l~g/ml)"1

Histone

Fig. 6. (A) Effect of histone c o n c e n t r a t i o n on the activity of protein kinase in either the presence or absence of Io -6 M cyclic AMP. Conditions were as described u n d e r Methods except for the variation of the c o n c e n t r a t i o n of histone. The reaction m i x t u r e contained 8. 4 #g of protein f r o m the P-3oo fraction. (B) Double-reciprocal plot of the d a t a in Fig. 6A.

regardless of whether cyclic AMP was present or not. Fig. 6B is a double reciprocal plot of the same data. The apparent Km for histone (50 #g/ml) was not affected by cyclic AMP, whereas the V was increased by the cyclic nucleotide. Apparent Kin for cyclic AMP. In experiments in which the cyclic AMP concentration was varied, maximal stimulation was found at approximately 2.1o -6 M cyclic AMP (Fig. 7A). Increasing the concentration above 5.1o -4 M cyclic AMP

90

A

80 70 0

c

60 50 40

i

0

60 t Km=2'5X10-8

t, o

E o.

B

/

30 a

20 10 10 "9

I

I

10 -7

10 -5

~AM~IMI

__

4

I

2

0

2

4

6

8

10

10-3 E Q.

'J

X10-7 (M. 1 )

Fig. 7- (A) Effect of increasing concentration of cyclic AMP on the activity of s m o o t h muscle p r o t e i n kinase. Conditions were as described u n d e r Methods except for the v a r y i n g concentration of cyclic AMP. The reaction m i x t u r e contained 8. 4 ~g of protein f r o m the P-3oo fraction. (B) Double-reciprocal plot of the d a t a in Fig. 7 A.

275

SMOOTH MUSCLE PROTEIN KINASE

110

/

~

cGMP

100 90 8O

o

70

~: 6o

E

60

o

////Z

50 40 30

1//.,7"

so

4o

2 _

\<.M,

20

I

I

]

t

IO-9

10-7

10-5

10-3

Km=l.3XIO"5

E

3o

-

'°

c

A

~ Km=l'4XlO"5

~ . . . ~ +cAMP 0

I

I

I

1

I

I

[

2

4

6

8

10

12

14

__

X10-4 (M-1 ) Concentration

Cyclic

NucJeotide (M)

Fig. 8. Effect of concentrations of cyclic nucleotides on the activity of s m o o t h muscle proteir kinase. Conditions were as described u n d e r Methods except for the cyclic nucleotide and its con. centration. The reaction m i x t u r e contained 8. 4 #g of p r o t e i n f r o m the P-3oo fraction. The activit3 in the absence of cyclic nucleotides was b e t w e e n 8 and 2o pmoles of 8,p incorporated. Fig. 9. Effect of A T P concentration on the activity of s m o o t h muscle protein kinase. Condition,. were as described u n d e r Methods except for the v a r y i n g concentration of ATP. The reaction mixt u r e contained 8. 4 / , g of protein f r o m the P-3oo fraction.

resulted in a significant inhibition of activity. The stimulation b y cyclic AMP was approximately Io-fold; in other experiments the stimulation of phosphorylation by lO .6 M cyclic AMP varied from 3- to Io-fold. A double-reciprocal plot of the data (Fig. 7 B) indicates an apparent Km of 2.5" lO -8 M cyclic AMP. Specificity for cyclic nucleotides. Since cyclic GMP occurs naturally 25,~6 and since cyclic GMP-stimulated protein kinases have been reported 27, it was of interest to test whether or not cyclic nucleotides other than cyclic AMP stimulated the smooth muscle protein kinase. The data presented in Fig. 8 show that cyclic GMP stimulated the enzyme to full activity only if used at unphysiologically high concentrations. Cyclic IMP was almost as effective in stimulating the smooth muscle protein kinase as was cyclic AMP, and, in fact, the maximal activity was slightly higher than that achieved in the presence of cyclic AMP. Cyclic CMP and cyclic UMP stimulated the enzyme at very high concentrations. The 2',3'-AMP had no effect on the smooth muscle protein kinase. Apparent Km for A TP. The data presented in Fig. 9 indicate that the protein kinase has an apparent Km of 1.4. lO-5 M for A T P in either the presence or absence oJ cyclic AMP when histone served as the second substrate of the enzyme. Cyclic AMF significantly increased the V of the reaction. Specificity for nucleoside triphosphate. In experiments designed to determine iJ nucleoside triphosphates other t h a n ATP could serve as phosphate donors, the protein kinase and histone were incubated either with or without lO -6 M cyclic AMP and the standard concentration of IT-a~PIATP (3.3" lO-6 M). Other non-radioactive nucleosid(

276

H. SANDS et al,

TABLE

Ill

NUCLEOSIDE

TRIPHOSPHATE

SPECIFICITY

OF SMOOTH

MUSCLE

PROTI~IN KINASE

A s s a y s w e r e p e r f o r m e d a s d e s c r i b e d u n d e r E x p e r i m e n t a l P r o c e d u r e s . C o n t r o l c o n t a i n e d 3 . 3 " lO M [V-3~P~ATP o n l y . T h e s a m e c o n c e n t r a t i o n o f Ey-3~PIATP w a s e m p l o y e d in a d d i t i o n t o i o -4 M o f u n l a b e l e d n u c l e o s i d e t r i p h o s p h a t e . T h e r e a c t i o n m i x t u r e c o n t a i n e d 8. 4 l¢g o f p r o t e i n f r o m the P-3oo fraction.

`4 ddition

pmoles 32p incorporated cyclic A M P

Control CTP GTP 1 ;TI }

% of control

4- cyclic ,43,IP

14.8 14.2 14. 4 14. 3

cyclic `4d!/It)

114. 4 IO4. 7 92.9 89,6

4- cyclic , q 3 I P -91.5 81.2 78.3

95 .6 96.8 96.6

lOO o

60 o.

-~

4O

2O 0

t

p

1

I

4

8

12

16

t ~'-7--

20

t

24

28

*

32

Mg +2 (raM)

F i g . IO. E f f e c t o f M g 2~ c o n c e n t r a t i o n o n t h e a c t i v i t y o f s m o o t h m u s c l e p r o t e i n k i n a s e in e i t h e r the p r e s e n c e o r a b s e n c e o f i o -~ M c y c l i c A M P . C o n d i t i o n s w e r e a s d e s c r i b e d u n d e r M e t h o d s e x c e p t f o r t h e v a r i a t i o n in M g 2+ c o n c e n t r a t i o n . T h e r e a c t i o n m i x t u r e c o n t a i n e d 8. 4 / ~ g o f p r o t e i n f r o m the P-3oo fraction,

TABLE

IV

EFFECTS

OF METAL IONS ON SMOOTH MUSCLE PROTEIN

KINASE

IN EITHER

THE PRESENCE

OR ABSENCE

OF Io -6 M CYCLIC A M P C o n d i t i o n s w e r e a s d e s c r i b e d u n d e r E x p e r i m e n t a l P r o c e d u r e s e x c e p t f o r t h e v a r i a t i o n in t h e c o n c e n t r a t i o n o f i o n , a s i n d i c a t e d . 8. 4 l t g o f t h e P - 3 o o f r a c t i o n w e r e u s e d . D a t a a r e e x p r e s s e d a s p m o ] e s o f 32p i n c o r p o r a t e d p e r 7 m i n .

Metal ion

[on concentration 2.5 m M cyclic A M P

N o a d d i t i o n 0.2 M g 2~ 14.1 C a 2+ 0. 4 M n 2~ 5.2 Ba2+ o.7 Co 2+ 14.o Z n 2+ 0.8

Io m M 4- cyclic A M P 0.2 60-7 i. l 25. t 4.5 97.0 2.2

Ratio

-- cyclic .4 M P

4- cyclic ,4 M P

Ratio

1.0 4.3 2.9 4 .8 6. 4 6. 9 2. 7

17.3 0.6 4 -(} 1 .o 13.8 1.2

-66.2 .3.3 24.7 5.9 83.o 1.9

3.8 .5.7 5.4 5-9 6.o 1.6

SMOOTH MUSCLE PROTEIN KINASE

277

triphosphates were added singly at lO -4 M (Table III). None of the nucleoside triphosphates significantly inhibited 32p transfer from ATP; the protein kinase appeared to require ATP specifically. Requirement for divalent cations. Fig. IO shows the relationship between protein kinase activity and Mg 2+ concentration. An apparent Km of approximately 2.5 mM Mg 2+ was calculated from the data ; there was no effect of cyclic AMP on the apparent Km for Mg 2÷. Co2+ (Table IV) stimulated the protein kinase more effectively than did Mg 2+. Other divalent metal ions were less effective than Mg 2+, but still permitted stimulation by cyclic AMP. Fig. I I shows that the observed stimulatory effect of Mg 2+ on protein kinase activity resulted solely from a decrease in the Km of the enzyme for ATP. The maximal velocity remained constant when the Mg 2+ concentration was varied.

60

o

~

~° 40

~

Km=I.3XIO-S

~° 20

/ ~

_

-cAMP

~

K"="4x'°s

tAmP]

X10"4 (M-1)

Fig. i z. E f f e c t o f increasing c o n c e n t r a t i o n s o f M g ~ a n d A T P on p h o s p h o r y ] a t i o n o f histone b y s m o o t h m u s c l e p r o t e i n kinase. C o n d i t i o n s were as d e s c r i b e d u n d e r M e t h o d s e x c e p t for t h e va ri a t i o n in Mg a+ a n d A T P c o n c e n t r a t i o n . The r e a c t i o n m i x t u r e c o n t a i n e d 8. 4 t,g of p r o t e i n from t h e P-3oo fractio n,

Sedimentation of protein kinase on sucrose density gradient. The sedimentation behavior and molecular weight of the smooth muscle protein kinase were studied using a 5-22% sucrose gradient either with or without lO -8 M cyclic AMP. Fig. 12 illustrates the results of such a study. A single peak of protein kinase activity was found when no cyclic AMP was present in the gradient (Fig. I2a). The protein corresponding to this peak had a mol. wt of I44 500 based on calculations using horse hemoglobin (tool. wt 64 o o o ) a n d rabbit muscle lactate dehydrogenase (mol. wt 142 ooo) as standards. Cyclic AMP stimulated this protein kinase activity. Preincubation of the enzyme with cyclic AMP and ATP (with concentrations and conditions similar to those used in the assay) followed b y sucrose density centrifugation in the presence of lO -8 M cyclic AMP resulted in a shift in the molecular weight of the enzyme. A single peak of activity with a tool. wt of approximately 50 ooo was obtained and cyclic AMP did not stimulate this activity (Fig. i2b). Between 85 and lOO% of the enzymic activity applied to the gradient were recovered. Cyclic AMP binding activity was found in a region with a mean tool. wt of 88 500. If preincubation with cyclic AMP (and ATP) was omitted and the enzyme subjected to sucrose density centrifugation in the presence of lO -8 M cyclic AMP, an additional peak of protein kinase activity was found on the shoulder of the peak corresponding to a tool. wt of

27 8

~.

SANDS et al.

A. No cAMP in Gradient LDH

10 8

Hb

;/

6

\

• 4

k

o---o +cAMP

0"

:

--

-cAMP

D 2 0 10

B. 10--6M cAMP in Gradient ~;~ LDH .~.%,.j \~b :

8

\

2000

~

?

1600

6

z e~

t

/

4

400

2 bottom

1

2

3

4

top

ml

Fig. 12. S e d i m e n t a t i o n of s m o o t h muscle protein kinase in a sucrose gradient. Sedimentation conditions were as described u n d e r Methods. (A) No cyclic AMP present; (B) p r e i n c u b a t i o n at enzyme with cyclic AMP and A T P at the concentration and conditions of the protein kinase assay; io -e M cyclic AMP in the gradient (similar findings were obtained w h e n the p r e i n c u b a t i o n with cyclic AMP was carried out in the absence of ATP). 0 - - - 0 , assayed in the presence of IO-~ M cyclic AMP; O---O, assayed in the absence of cyclic AMP; A - - A , cyclic[SH]AMP binding. Hb, horse hemoglobin; L D H , r a b b i t muscle lactate dehydrogenase.

5 ° ooo. The activity in this peak (mol. wt greater than 5o ooo) was stimulated by the addition of cyclic AMP to the kinase assay and was observed in several experiments. The location of the cyclic AMP binding activity within the gradient was the same as that shown for the preincubated enzyme. DISCUSSION

The widespread distribution of cyclic AMP-dependent protein kinases led Kuo and Greengard la to propose that the effects of cyclic AMP in general might be mediated by protein kinases. The role of protein kinase in the activation of glycogen phosphorylase of skeletal muscle is well established ~s. The activities of glycogen synthetase 9 and of the triglyceride lipase of adipose tissue 2° are also regulated by the phosphorylation of the respective enzymes catalyzed by cyclic AMP-stimulated protein kinases. It has been suggested that the regulation of transcription in eukaryotes 3°, of synaptic transmission 31, of vasopressin-induced changes in the permeability of the medullary membranes of the kidney 32, of the action of hypophysiotropic hormones TM, the activity of the lacrymal gland ~3, regulation of the activity of the mammary gland ~4, and the control of diurnal rhythm via the pineal gland 15 are mediated by protein kinases. In this paper we describe certain properties of a partially purified protein kinase isolated from bovine bronchial and tracheal smooth muscle. The procedure which we employed led to the isolation of one major species of

SMOOTH MUSCLE PROTEIN KINASE

279

protein kinase (a small secondary peak of kinase activity was seen after DE-52 column chromatography of several preparations). This is in contrast with findings made employing the pituitary 16 and skeletal muscle ~6 as sources of protein kinase. In the case of the pituitary, cyclic AMP-dependent and cyclic AMP-independent forms were observed after SE-Sephadex C 5 ° chromatography; whereas in the case of skeletal muscle two large and distinct peaks of cyclic AMP-dependent protein kinase activity were found after DE-52 chromatography. Multiple forms of rat skeletal muscle protein kinase have been reported recentlyaL It is of interest to compare some of the properties of the bovine tracheal smooth muscle protein kinase with those of protein kinases from other tissues. Although the assay conditions differed, the p H optimum of 6.5 to 7 reported here for smooth muscle protein kinase is similar to that of the enzyme from the pituitary 16, brain 36, and skeletal muscle 36, and differs from the p H optimum of 8.5 to 9.o reported for rabbit reticuloeytes 39, that of frog bladder epithelium 4° of p H 7.5, and trout testes 41 also of p H 7.5. Stimulation of the tracheal smooth muscle protein kinase was maximal at a cyclic AMP concentration of lO .6 M and the apparent Km for cyclic AMP was 2.5" IO-s M. Similar Km values were reported for the protein kinases of the pituitary 16, skeletal muscle a6, and other tissues 1~. Protein kinases obtained from the brain, adrenal, ovary, stomach, and duodenum had apparent Km values of I . I .IO 7-1.6. lO _7 cyclic AMP ~2. The stimulation of protein kinase activity by high concentrations of cyclic nucleotides other than cyclic AMP observed in the present study has also been reported for other protein kinases'6,a6, 36. Cyclic GMP, which occurs naturally, stimulated the activity of the bovine tracheal smooth muscle protein kinase only at unphysiologically high concentrations. A smooth muscle protein kinase stimulated by physiological concentrations of cyclic GMP occurs in lobster smooth muscle and various tissues of other arthropods 17. The bovine tracheal smooth muscle protein kinase appears to be specific for ATP as donor of the y-phosphate; other nucleoside triphosphates did not inhibit the transfer of phosphate from ATP. The apparent Km of the protein kinase for ATP was 1.4. io 5 M in the presence of IO mM Mg2+ and was not affected by cyclic AMP. As the Mg 2+ concentration was lowered to I raM, the apparent Km increased, whereas the V was unaffected. Similar findings, and an explanation for them, have been reported by Reimann et al. 36 for the protein kinase of skeletal muscle. In the case of the bovine pituitary protein kinase, lowering the Mg ~÷ concentration affected both the Km for ATP and the V of the reaction 16. The apparent Km of 1.4. IO 5 M ATP determined in the present study for the protein kinase of bovine tracheal smooth muscle was unaffected by cyclic AMP, and is similar to the Km ATe reported for several other tissues and speciesl6, 36. The protein kinase of bovine brain 36 which had an apparent Km of 1. 3 • lO .4 M in the presence of cyclic AMP and of 2.2. io -4 M in its absence, and the protein kinase of rabbit reticuloeyte 39 which showed a Km of 1. 7"1o -4 M in the presence and 2.5" lO -4 M in the absence of cyclic AMP, seem to be exceptional. Our data show that in the case of bovine tracheal smooth muscle protein kinase, cyclic AMP activates b y increasing the V rather than b y affecting the Km for the substrates of the reaction. Studies of the protein kinase of rabbit reticulocytes 42 as well as of other species and tissuesl6, 36 suggest that the enzyme is a dimer composed of a catalytic subunit (mol. wt of 4 ° ooo-6o ooo) and of an inhibitory subunit (mol. wt of approximately

28o

H. SANDS gt al.

8o ooo). The d i m e r is i n a c t i v e ; the b i n d i n g of cyclic A M P to t h e i n h i b i t o r y s u b u n i t causes it to dissociate from the c a t a l y t i c s u b u n i t which becomes e n z y m i c a l l y active. I n the case of t h e bovine t r a c h e a l s m o o t h muscle p r o t e i n kinase a single p e a k of cyclic A M P - d e p e n d e n t a c t i v i t y occurred when the p r e p a r a t i o n was s e d i m e n t e d in the absence of cyclic A M P (Fig. I2a); the mol. wt of this fraction was a p p r o x i m a t e l y 144 5oo. W h e n the p r e p a r a t i o n was i n c u b a t e d with cyclic A M P prior to s e d i m e n t a t i o n , a peak of cyclic A M P - i n d e p e n d e n t p r o t e i n kinase a c t i v i t y of mol. wt 5o ooo a n d a p e a k of cyclic AlVIP-binding a c t i v i t y of a tool. wt of a p p r o x i m a t e l y 88 5oo were found. No a t t e m p t was m a d e to r e c o n s t i t u t e the cyclic A M P - d e p e n d e n t p r o t e i n kinase by r e c o m b i n a t i o n of the c a t a l y t i c a n d r e g u l a t o r y components. I t appears, however, t h a t unless the e n z y m e is p r e i n c u b a t e d w i t h cyclic AMP, complete dissociation does not occur. I n the absence of p r e i n c u b a t i o n with cyclic A M P a p e a k of kinase a c t i v i t y slightly heavier t h a n the 5 ° ooo mol. wt e n z y m e was found ; the p r o t e i n of this fraction showed cyclic A M P - s t i m u l a t e d p r o t e i n kinase a c t i v i t y . The 50 ooo mol. wt cyclic A M P - i n d e p e n d e n t c a t a l y t i c unit a n d the cyclic A M P b i n d i n g a c t i v i t y in the region of 85 ooo mol. wt were also f o r m e d u n d e r these conditions. The significance of this o b s e r v a t i o n is not clear. A similar finding, however, was m a d e b y M i y a m o t o et al. ~ w i t h respect to the b e h a v i o r of bovine b r a i n p r o t e i n kinase. The cyclic A M P - s t i m u l a t e d form of t h a t e n z y m e h a d a mol. wt of I4o ooo. W h e n it was centrifuged t h r o u g h a g r a d i e n t c o n t a i n i n g either cyclic A M P or histone, a cyclic A M P - s t i m u l a t e d c o m p o n e n t of mol. wt 8o ooo was formed. A cyclic A M P - i n d e p e n d e n t form of the e n z y m e occurred, when the g r a d i e n t c o n t a i n e d both cyclic A M P a n d histone. B o t h the 14o ooo and 8o ooo mol. wt p r o t e i n s b o u n d cyclic AMP. The occurrence of a cyclic A M P - d e p e n d e n t p r o t e i n kinase in t r a c h e a l smooth muscle suggests t h a t the e n z y m e m a y p l a y a role in the r e l a x a t i o n of the muscle b r o u g h t a b o u t b y cyclic AMP. An a t t e m p t to i d e n t i f y the endogenous substrate(s) oI the e n z y m e in s m o o t h muscle is now in progress in this l a b o r a t o r y . ACKNOWLEDGMENTS This w o r k was s u p p o r t e d b y Research G r a n t s R R - o 5 4 7 4 - i o a n d H L - I 4 9 6 4 - o I from the N a t i o n a l I n s t i t u t e s of H e a l t h , U n i t e d S t a t e s Public H e a l t h Service and R e s e a r c h G r a n t GB 8292 from the N a t i o n a l Science F o u n d a t i o n . REFERENCES I 2 3 4 5 6 7 8 9 IO i1 I2

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SMOOTH MUSCLE PROTEIN KINASE 13 14 15 16 17 i8 19 20 2i 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43

281

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